Two fundamental virologic issues represent major hurdles in the development of effective antiretroviral therapy: quantitation of viral load and drug resistance. The aims of this proposal are to address each of these two issues in the laboratory by applying newly developed methods to clinical material from subjects on ACTG study protocols. In the area of quantitation, we propose to develop and optimize assays for viral infectivity, for viral DNA and RNA by PCR and for viral RNA by the self sustained sequence reaction (3SR) methodology. These methods will be applied to specific components of the peripheral blood including plasma, CD4 lymphocytes, monocyte/macrophages and dendritic cells. The quantitation of viral load, and the relative amounts of integrated and unintegrated DNA, in these blood components will be correlated with disease stage and antiretroviral therapy. In the area of drug resistance, methods that are being developed and optimized to assay for drug susceptibility of isolates of HIV and for mutations characteristic of drug resistance will be systematically applied to ACTG protocols. Only with precise and relevant markers of viral endpoints can reliable determinations be made of the impact of antiretroviral intervention.